1. Field of the Invention
The present invention relates to a xylitol producing microorganism introduced with arabinose metabolic pathway and a method for producing xylitol efficiently by using the same, more precisely a xylitol producing microorganism introduced with arabinose metabolic pathway to inhibit the generation of arabitol that interrupts the purification and crystallization of xylitol and at the same time to utilize arabinose for cell growth, and a production method of xylitol with high productivity by using the same.
2. Description of the Related Art
Xylitol is the pentose sugar alcohol having high sweetness, which thus has been widely used as a functional sweetener that can take the place of sugar. Xylitol has the sweetness similar to sugar, but its metabolic process in human body is not related with insulin. Therefore, xylitol has been used as an alternative sweetener of sugar for diabetics. In particular, xylitol has the activity of inhibiting the growth of Streptococcus mutans, the cariogenic bacteria, so that it is also widely used as an anticariogenic material.
Xylitol is produced by chemical-reducing hemicellulose hydrolysates containing lots of xylose, such as corncob and sugar cane stalk. However, such chemical method does not favor the separation and purification of xylose from other pentose and hexose such as arabinose and glucose, etc, and has disadvantages of high cost for the separation and purification and as low xylose recovery rate as 50˜60%. In addition, such chemical method is also characterized by high temperature/high pressure procedure using hydrogen gas and nickel catalyst, delivering issues of high risk and environmental problem.
To overcome the above disadvantages or problems of the conventional chemical method, a biological approach has been actively made to produce xylitol. Unlike the chemical method, the biological method can utilize xylose even with low purity as a raw material and the procedure itself can be completed at room temperature under normal pressure, suggesting that the biological method is pro-environmental production process. To produce xylitol with high productivity and high yield using the biological method, various bacteria, yeast, fungi, recombinant yeast, etc, have been studied (Winkelhausen, E. et al., J. Ferment. Bioeng. 86:1-14, 1998; Granstrom, T. B. et al., Appl. Microbiol. Biotechnol. 74:277-281, 2007). However, bacteria and recombinant yeast have been confirmed not to be appropriate for the industrial production of xylitol because their xylose metabolic pathways are too weak or not very efficient. On the other hand, among many yeast/fungi, Candida sp. strains show high capacity to utilize xylose, compared with other microorganisms, making them promising candidates for the biological production of xylitol with high productivity and high yield.
According to the previous studies, Candida sp. strains such as C. guillermondi, C. parapsilosis, and C. tropicalis can convert xylose introduced from outside of the cell into xylitol with the help of xylose reductase, and further can convert the produced xylitol into xylulose with the help of xylitol dehydrogenase. The xylulose can be further converted into xylulose-5-phosphate by xylulokinase and be consumed for the cell growth and maintenance via pentose phosphate pathway (Laplace, J. M. et al., Appl. Microbiol. Biotechnol., 36:158-162, 1991; Hahn-Hagerdal, B. et al., Enzyme Microb. Technol., 16:933-943, 1994).
At this time, the xylose reductase uses NADPH (nicotinamide adenine dinucleotide phosphate) as a cofactor, and the xylitol dehydrogenase uses NAD+ (nicotinamide adenine dinucleotide) as a cofactor. Xylitol converted from xylose mediated by xylose reductase is converted again into xylulose by xylitol dehydrogenase. At this time, if oxygen supply is limited in a medium to make the concentration of dissolved oxygen to be 0.5%˜2.0%, intracellular redox imbalance is induced, which means NAD+, the cofactor needed by xylitol dehydrogenase, becomes short, resulting in the inhibition of the conversion of xylitol into xylulose. As a result, xylitol is accumulated in cells and medium, indicating that xylitol is produced from xylose with the yield of 50˜60%. That is, in the conventional method to produce xylitol by using a xylitol producing microorganism, it is necessary to regulate dissolved oxygen to the degree of low concentration by limiting oxygen supply (limited aeration). So, studies have been actively undergoing to increase xylitol productivity with high yield by inducing intentional imbalance of oxidation reduction potential in cells by maintaining dissolved oxygen concentration low by limited aeration (Kim, S. Y. et al., J. Ferment. Bioeng., 83(3):267-270, 1997; Korean Patent No. 1996-030577).
Korean Patent No. 10-0169061 describes a method for producing xylitol by using concentrated Candida parapsilosis strain with regulating the concentration of dissolved oxygen to 0.8˜1.2%. However, it is actually almost impossible to regulate the concentration of dissolved oxygen as low as the above when xylitol is produced in a large industrial scale by using a large volume fermenter and if possible, the yield cannot exceed 50˜60%. Korean Patent No. 10-0259470 describes a stirring speed to regulate oxygen level in a medium under 1% DOT (oxygen concentration in a medium presented as %). However, there is still inconvenience in the process. Korean Patent Application No. 95-37516 describes a production method of xylitol by using a transformant strain of Candida parapsilosis, in which xylitol production optimization achieved by the regulation of oxygen partial pressure in the medium is described. Korean Patent Application No. 96-13638 describes the optimum medium and culture conditions for the production of xylitol from the said transformant strain. However, in this method, the yield of xylitol was not more than 70% even under the optimum conditions. The present inventors have developed a Candida tropicalis transformant in which xylitol dehydrogenase activity is completely inactivated, based on the fact that xylitol produced in Candida sp. strain is converted into xylulose by xylitol dehydrogenase (Korean Patent No. 10-0730315). According to the previous methods, limited aeration in the medium was necessary to induce intracellular oxidation reduction potential imbalance, in order to inhibit the activity of xylitol dehydrogenase converting xylitol into xylulose. However, in the transformant in which xylitol dehydrogenase is inactivated, xylitol produced from xylulose is not used for cell growth anymore, suggesting that oxidation reduction potential imbalance is not needed. By this method, xylose can be converted into xylitol with the yield of 97-98%.
Even though the yield of xylitol from xylose was maximized, there is another problem to solve, which is that biomass hydrolysate, used as the raw material for the production of xylitol, contains not only xylose but also a huge amount of arabinose. Xylose reductase in the xylitol producing microorganism affects arabinose as well. So, when the biomass hydrolysate is directly used as a law material, arabitol is also produced. Arabitol, the pentose sugar alcohol like xylitol, has similar molecular structure and physical properties to xylitol. It is thus unwanted byproduct that lowers production rate of xylitol by interrupting purification and crystallization of xylitol. Therefore, when xylose/arabinose mixed medium is used for the production of xylitol, a novel technique to produce xylitol without producing arabitol is required.
The present inventors performed codon optimization to express efficiently the arabinose metabolic pathway involved enzymes such as L-arabinose isomerase (araA), L-ribulokinase (araB), and L-ribulose-5-phosphate 4-epimerase (araD) in Candida sp. Then, each gene was inserted in the cassette containing glyceraldehyde-3-phosphate dehydrogenase promoter and URA3, the selection marker. The prepared cassette was introduced in Candida sp. As a result, the present inventors completed this invention by confirming that xylitol can be produced with high productivity by inhibiting the production of arabitol interrupting the purification and crystallization of xylitol.